A DNA chip is a rigid flat surface, typically glass or silicon, with short chains of related nucleic acids spotted in rows and columns on it. Hybridization between a fluorescently-labeled DNA and specific locations on the chip can be detected and analyzed by computer-based instrumentation. The information derived from the results of hybridization to DNA chips is stimulating advances in drug development, gene discovery, gene therapy, gene expression, genetic counseling, and plant biotechnology.
Among the technologies for creating DNA chips are photolithograpy, xe2x80x9con-chipxe2x80x9d synthesis, piezoelectric printing, and direct printing. Chip dimensions, the number of sites of DNA deposition (sometimes termed xe2x80x9caddressesxe2x80x9d) per chip, and the width of the DNA spot per xe2x80x9caddressxe2x80x9d are dependent upon the technologies employed for deposition. The most commonly used technologies produce spots with diameters of 50-300 xcexcm. Photolithography produces spots that can have diameters as small as 1 micron. Technologies for making such chips are known to those skilled in these arts and arc described, for instance, in U.S. Pat. Nos. 5,925,525, 5,919,523, 5,837,832, and 5,744,305, which are incorporated herein by reference.
Hybridization to DNA chips can be monitored by fluorescence optics, by radioisotope detection, and by mass spectrometry. The most widely-used method for detection of hybridization employs fluorescently-labeled DNA, and a computerized system featuring a confocal fluorescence microscope (or an epifluorescence microscope), a movable microscope stage, and DNA detection software. Technical characteristics of these microscope systems are described in U.S. Pat. Nos., 5,293,563, 5,459,325, and 5,552,928, which are incorporated herein by reference. Further descriptions of imaging fluorescently immobilized biomolecules and analysis of the images are set forth in U.S. Pat. Nos. 5,874,219, 5,871,628, 5,834,758, 5,631,734, 5,578,832, 5,552,322, and 5,556,539 which are incorporated herein by reference.
In brief, these conventional approaches to visualizing the surface of a DNA chip involve placing the chip on a microscope stage, moving the stage to put the sample into focus with a microscope objective, and triggering a digital camera or similar device to capture an image. An objective is a device made of a group of lenses that have a sophisticated design that collects light from the sample, magnifies the image of the sample, and minimizes the unavoidable image and color distortion caused by the passage of the light through the objective. The light-collected from the sample passes through the objective and through a set of mirrors and lenses until is delivered to an eyepiece or the camera. The light path is the path that the light takes from the point where it leaves the surface of the sample until it reaches an imaging device such as an eyepiece or camera. The microscopes are integral with light sources that direct light on to the sample.
These microscopes also have sets of optical filters that allow for viewing of fluorescent images. The DNA that is hybridized to the surface of the DNA chip is typically labeled with fluorescent molecules that absorb light at one wavelength and then emit a different wavelength. The microscope is equipped with sets of optical filters that block the wavelengths of light from the light source but allow the light emitted by the fluorescent molecules to pass through the light path to reach the eyepiece or camera. The light source is typically integral with the microscope and is an important part of the imaging system.
These conventional microscopes are sophisticated and expensive instruments that require training and maintenance. A single microscope objective typically has multiple lenses. A lens, as used herein, means a transparent solid material shaped to magnify, reduce, or redirect light rays. A light filter or mirror is distinct from a lens. Furthermore, use of a microscope requires a dedicated workspace that is approximately the size of a typical desk. Conventional microscopes have a light path that is several centimeters long that transmits the collected light through air and other assorted optical devices within the light path. One of the challenges in microscopy is making the microscope as efficient as possible in capturing all of the light that leaves the sample surface so that an optimal image may be made.
The costly instrumentation conventionally used to image DNA clips impedes the broad usage of DNA chip technologies.
What is needed is an inexpensive, low-maintenance alternative spot detection method for DNA chip analysis that is easy to use and requires a minimum of space and maintenance.
Integrated electronic circuit arrays for light-detection (herein referred to as members of the group of detectors called electronic light detector arrays) and analysis are readily available. They generally are based on CCD (charge-coupled device) or CMOS (complementary metal oxide semiconductor) technologies. Both CCD and CMOS imaging detectors are two-dimensional arrays of electronic light sensors. Each array consists of a set of known, unique positions that are also called addresses. Each address in a CCD or CMOS array is occupied by a sensor that covers an area that is typically shaped as a box or a rectangle; this area or the area occupied by a single sensor is referred to as a pixel. Herein, a light-detecting sensor located on a pixel is called a detector pixel. A detector pixel may be in a CCD sensor, a CMOS sensor, or other device that detects or measures light. The sizes of detector pixels vary widely and may have a diameter or length of 0.2 xcexcm, which is the theoretical limit of resolution of the light microscope. Thus an invention that directly employs electronic detection instead of a conventional optical system is potentially as powerful as any light microscope. Light, as used herein, means any electromagnetic emission of at least 120 nm wavelength and includes ultraviolet, visible, and infrared light.
CCDs, widely used in consumer and scientific applications such as digital recorders and digital cameras, are most sensitive, and may be made with detector pixels that are smaller than those of CMOS devices. CMOS devices are now beginning to be incorporated in recorders and cameras because they are less expensive to produce. CMOS devices also are easier to interface with external control systems than CCDs. Some readily-available CMOS devices are capable of acquiring, digitizing, and transmitting an image without additional circuitry, while CCD arrays require two or more additional circuit elements to accomplish the same tasks.
The present invention describes an inexpensive device and method for resolving the light spots emitted by a light-transparent DNA chip. The method is direct mapping of the light emitted by a single DNA spot onto corresponding detector pixels of an electronic light detector array system. One method is to put the DNA chip in direct physical contact with the electronic light detector array system. In a modification of this basic method, a simple optical system, such as a single mapping lens, maps an enlarged or reduced version of the DNA array onto the electronic light detector array. Computer software processes the data from the electronic light detector array system. The data may be treated as a two-dimensional map or otherwise processed as an array.
Implementation of the described method would replace the expensive optical detection systems currently employed for DNA chip analysis with an inexpensive system. This system comprises an electronic light detector array, a filter, and, optionally, a mapping lens system. The invention enables the DNA chip to be mapped onto the electronic light detector array. Thus each position on the DNA chip surface has a corresponding position or set of positions on the detector array whereby a fluorescence at an address on the DNA chip surface is projected onto a known pixel or set of pixels.
Direct mapping is inexpensive. It eliminates the need for a complicated microscope that requires maintenance and trained personnel. It captures light directly from the sample; by eliminating many lenses, disadvantages stemming from use of many lenses are reduced. Direct mapping enables direct capture of light so that a maximal amount of light is captured from the sample; minimizing the loss of light creates a very sensitive imaging system.
Electronic light detector array systems include an electronic light detector array such as a CMOS or CCD chip and the associated equipment for visualization. This associated equipment includes filters, lenses, and light sources. The filters may be any filter used to reflect or selectively pass or reject light wavelengths. Such filters include edge filters, narrow band filters, dichroic mirrors and filters used in the visualization arts, including optical, ultraviolet, confocal, and two- or multi-photon microscopy. Light sources include those commonly used in the visualization arts, including optical, ultraviolet, confocal, and two-photon or multi-photon microscopy. Light sources further include light lamps and light lasers, such as visible-light lamps, ultraviolet lamps, mercury lamps, and lasers, including argon lasers, helium-cadmium lasers, semiconductor lasers, and so forth.
Various associated equipment is found in association with these devices, and are known to those skilled in these arts. Such equipment includes manual or electric filter-switchers, movable mirrors, and motors and controls to raster a laser across a sample. Various equipment is associated with CCD and CMOS sensors, which are incorporated into a myriad of commercially available cameras and detectors. For instance, various equipment and techniques are known for producing a color image using red, green, and blue detection; for example, an image may be split into three images, each of which is sent through a red, green, or blue filter to a CCD sensor. Or CCD sensors may be placed on a chip with different sensitivities to red, green, and blue light. Such equipment include techniques and electronic means for improving an image, and include electronic filters (high-pass, low-pass. etc.), time and frame-averaging, image subtraction, and other techniques known to those skilled in these arts.
The detection system may be configured to excite, detect, filter, and process fluorescence from conventional fluorophores, for example, fluorophores described in catalogues published by Life Technologies, Inc. (Rockville, Md.), Sigma-Aldrich, Inc. (SIGMA, ALDRICH, and FLUKA brand names; St. Louis, Mo.), Pierce Chemicals (Rockford, Ill.), and other suppliers known to those skilled in these arts. Similarly, other DNA visualization techniques are currently known and used, and many examples of these technologies are set forth in these same sources. For instance, calorimetric systems that create a color in the visible light wavelength, for instance those based on a stain or on enzyme activity, may be adapted to visualize DNA. And amplification systems that may be used in combination with a colorimetric or fluorescent system may also be used; for example, avidin-biotin or antibody-based techniques. For example, the target DNA labeled with biotin may be placed on the DNA chip. After a washing protocol is performed, the sample may be exposed to labeled avidin, which makes a strong bond to the biotin. The label on the avidin may be a fluorophore (or an enzyme such as horseradish peroxidase (HRP) that is suitable for calorimetric assay). Additionally, DNA may be labeled by chemiluminescence or chemifluorescence and subsequently detected.
DNA may be attached to substrates that pass light by a variety of means known to those skilled in these arts. For instance, glass or quartz may be treated with silanes to create carboxyl or amine groups that may be used in further chemical reactions for immobilizing DNA. Such techniques and many others known to those skilled in these arts are included in the patents incorporated by reference, above, as well as in the following references, which are incorporated herein by reference: Laursen et al., xe2x80x9cSolid Phase Methods in Protein Sequence Analysis,xe2x80x9d in Methods of Biochemical Analysis, vol 26 John Wiley and Sons, Inc. 1980, pp. 202-215; xe2x80x9cImmobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarraysxe2x80x9d, Rogers Y H, Jiang-Baucom P, Huang Z J, Bogdanov V, Anderson S, Boyce-Jacino M T, Anal Biochem 1999 January 1;266(1):23-30; xe2x80x9cCovalent attachment of DNA oligonucleotides to glassxe2x80x9d, Cohen G, Deutsch J, Fineberg J, Levine A, Nucleic Acids Res 1997 February 15;25(4):911-2; xe2x80x9cHybridization of DNA targets to glass-tethered oligonucleotide probesxe2x80x9d, Beattie W G, Meng L, Turner S L, Varma R S, Dao D D, Beattie K L, Mol Biotechnol 1995 December ;4(3):213-25; xe2x80x9cPreparation of glass plates with cerium oxide for DNA sequencingxe2x80x9d, Millard D, de Couet H G, Biotechniques 1995 October; 19(4):576; xe2x80x9cBiologically Functional Materialsxe2x80x9d, Allan S. Hoffmnan, in Biomaterials Science, B. D. Ratner, A. S. Hoffmnan, F. J. Schoen, and J. E. Lemons, Eds., pp. 124-130.
The invention may be used with DNA or with other combinations of hybridizable molecules, for instance RNA-DNA or DNA-protein interactions. DNA-DNA hybridization has been used as an example but the invention includes all polynucleic acid hybridization techniques, including RNA-RNA hybridization. Polynucleic acid, as used herein, means DNA, RNA, two or more oligonucleotides or oligonucleosides, and all long or short sequences of nucleic acids. xe2x80x9cDNA chipxe2x80x9d or xe2x80x9cpolynucleic acid chipxe2x80x9d as used herein refers not only to DNA sequences immnobilized on small solid substrates, but also refers to RNA, etc., and generally to a device with biomolecules immobilized to it.
The present invention is a device and method for detecting the pattern of polynucleic acid hybridization to a surface. The device includes (a) a positioning device for receiving a nucleic acid chip and keeping the chip in a sampling position, the nucleic acid chip being an object with a flat sample surface and an opposed surface that is joined to the sample surface by a thickness, with the sample surface having sequences of nucleic acids immobilized thereto, with each sequence being immobilized to a particular chip address. And, (b) an electronic light detector array, the detector array comprising detector pixels, the detector pixels being sensors located at particular detector pixel addresses, wherein the sampling position places the sample surface of the chip at a well-defined position relative to the electronic light detector array so that light leaving a chip address is substantially directed onto at least one detector pixel with an address that is correlated to the chip address.